human genome wide crispra sgrna library  (Addgene inc)

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: (A) Summary of gene deletion vs. gene overexpression approaches. (B) Rationale for the present study. (C) Experimental outline of the present study. (D) HEK293 and Jurkat cells were inoculated with different volumes of Ebola or rabies pseudovirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This revealed that Jurkat cells are largely refractory to Ebola or rabies pseudovirus entry, relative to HEK293 cells. (E) Construction of a clonal Jurkat cell line, known as “Jurkat C6”, stably expressing a degron-tagged CRISPRa construct ( left ). Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence or absence of TMP (1 μM) for 3 days, followed by flow cytometry to detect hCD19 expression ( right ). (F) At each of the indicated points of the genome-wide CRISPRa screen, the cell population was challenged with Ebola or rabies pseudovirus, and cell infectivity was evaluated by flow cytometry. Upon successive rounds of the screen, the cell population became progressively more susceptible to infection to either Ebola or rabies pseudovirus, respectively. (G) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for Ebola pseudovirus entry. (H) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for rabies pseudovirus entry.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Over Expression, Marker, Mutagenesis, Flow Cytometry, Infection, Stable Transfection, Expressing, Construct, Transduction, Genome Wide

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) Plasmids used to pseudotype non-replicating lentiviruses with either Ebola or rabies envelope proteins. EBOV-GP: glycoprotein of Ebola virus, Makona variant. RABV-GP N2C: glycoprotein of rabies virus, N2C variant. B) HEK293 and Jurkat cells were inoculated with different volumes of VSV envelope protein-pseudotyped lentivirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This served as a positive control to confirm that Jurkat cells and HEK293 cells are both susceptible to VSV pseudovirus entry. C) Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence of different TMP concentrations (0-4 μM) for 2-3 days, followed by flow cytometry to detect human CD19 expression.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Virus, Variant Assay, Marker, Mutagenesis, Flow Cytometry, Infection, Positive Control, Transduction, Expressing

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. B) L-SIGN or DC-SIGN were expressed in Jurkat or primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of primary T cells to authentic Ebola and Sudan virus infection.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Expressing, Control, Flow Cytometry, Infection, Mutagenesis, Virus

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. As positive controls, flow cytometry was used to confirm successful delivery of the sgRNA construct as part of the CRISPRa workflow (as denoted by BFP expression) and that NGFR was expressed (upon cDNA expression). B) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. Cells expressing the highest levels of L-SIGN and DC-SIGN were preferentially infected by Ebola pseudovirus. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. On days 0, 1, and 2 post-infection, flow cytometry was performed to determine the percentage of infected cells and qPCR was performed on cell culture supernatants to quantify viral genome replication. This revealed that L-SIGN or DC-SIGN expression enabled authentic Ebola and Sudan virus entry into primary human T cells, but viral genome replication was impaired, perhaps reflective of cell-intrinsic restriction factors.

Article Snippet: The human genome-wide CRISPRa sgRNA library (hCRISPRa-v2 [Addgene, 1000000091]) comprises 209,080 sgRNAs targeting 18,915 human genes (approximately 10 sgRNAs per gene), in addition to 3,790 non-targeting control sgRNAs .

Techniques: Expressing, Control, Flow Cytometry, Infection, Construct, Mutagenesis, Virus, Cell Culture

Journal: bioRxiv

Article Title: Antibody-Dependent Heterotypic Syncytia Drive COVID-19 Inflammation and Disease Progression

doi: 10.64898/2026.02.11.705426

Figure Lengend Snippet: A , Schematic of the CRISPR-Cas9 screen. B , Volcano plot of genes from the screen, ranked by fold change and adjusted P value. C , Representative flow cytometry dot plots of GW01-induced syncytia between A549-spike and FCER1G -KO (FcRγ-KO) THP-1 clones versus non-targeting control (NT), and quantification of syncytia(right inset)(mean ± SEM, n=3, * P <0.05, **** P <0.0001). D , Representative plots A549-spike fusion with ADAM10-KO THP-1 clones versus control and quantification of syncytia. E , Rescue of FcRγ: representative plots for FcRγ-KO-LVX (empty lentiviral vector), FcRγ-KO-FcRγ (FcRγ cDNA), and NT-LVX; with quantification of syncythia. F , Rescue of ADAM10: representative plots for ADAM10-KO-LVX (empty vector), ADAM10-KO-ADAM10 (ADAM10 cDNA), and NT-LVX, along with quantification of syncytia. G , Effect of CD64 or CD32 blocking antibodies on GW01-dependent syncytium formation measured by NanoLuc assay. H , Effect of the ADAM10 inhibitor GI254023X on GW01-dependent syncytium formation. I , Representative fluorescence images of syncytia (yellow merge) between THP-1-mCherry (red) and A549-spike (green; CFSE) at 3 hour post co-culture, with or without GI254023X. Scale bar, 100 μm. Data are presented as mean ± SEM, n=3, * P <0.05, *** P <0.001, **** P <0.0001 by one-way ANOVA.

Article Snippet: The genome-wide CRISPR sgRNA library (Addgene, 101926-101934) was packaged into lentiviruses.

Techniques: CRISPR, Flow Cytometry, Clone Assay, Control, Plasmid Preparation, Blocking Assay, Fluorescence, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: CDK4/6 inhibition overcomes venetoclax resistance mechanisms with enhanced combination activity in acute myeloid leukemia

doi: 10.1016/j.xcrm.2025.102526

Figure Lengend Snippet: Combining ven+palbo mitigates single-agent resistance due to clinically observed mutations (A) Enrichment of individual sgRNAs for RB1, BAX, and IKZF1 shown as fold change over DMSO control following a 21-day exposure to palbo, ven, or ven+palbo in OCI-AML2 Cas9 C6 cells. (B) Immunoblot showing efficiency of knockdown of RB1, BAX, and IKZF1 proteins in OCI-AML2 cell lines. A cell line expressing an NT sgRNA was used to generate a control cell line. Vinculin was used as a protein loading control. Par, parental; NT, non-targeting. (C–F) Dose-response curves for OCI-AML2 NT and KO cell lines evaluated for drug sensitivity to palbo, ven, or the combination. Data points denote the mean normalized cell viability ± SD for 3 replicates. (G) IC 50 values derived from dose-response curves of OCI-AML2 cell line drug sensitivity assays shown in (C)–(F). Data represent the mean IC 50 ± SD for 3 replicates (∗ p ≤ 0.05 and ∗∗ p ≤ 0.01 by Student’s t test). (H–J) Outgrowth of OCI-AML2 Non-targeting (H), OCI-AML2 Bax KO (I), and OCI-AML3 cell lines (J) treated with palbo, aza, and ven single agents, duplicate combinations and the triplet. Total viable cells over a 14-day drug treatment are shown. Data points denote the mean total number of viable cells ± SD for 3 replicates. One-way ANOVA with Tukey’s post-test for multiple comparisons was used for day 7 and day 14 time points as indicated. (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001) (K) Immunoblot of apoptotic proteins in OCI-AML2 cells, drug treated for 14 days.

Article Snippet: Clonal OCI-AML2 Cas9 C6 cells were used for genome wide knockout with sgRNA library (Addgene #67989) as described above.

Techniques: Control, Western Blot, Knockdown, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Ribosome-associated quality control of aberrant protein production during amino acid limitation

doi: 10.64898/2026.01.14.699605

Figure Lengend Snippet: (A) A dual reporter system with a Flag-mCherry CGG -4xCGG-DHFR CGG (internal control, no translation disruption) and a Flag-YFP CGG -4xAGA-DHFR CGG was integrated into the AAVS1 locus in cells expressing CRISPRi or CRISPRa machinery (see Methods) to query the effect of a genome-wide library of CRISPRi/a sgRNAs on specific translation disruption signal. Flow cytometry was used to sort high and low responding populations. (B-D) Total score (phenotype score * Mann-Whitney (M-W) p-value, see Methods) versus hit rank for highest (high YFP) and lowest (low YFP) scoring genes in K562 CRISPRi (B), 293T CRISPRa (C), and 293T CRISPRi (D) screens. Hits are labeled and colored by associated function. Dashed line marks score cut-off where FDR = 0.25. (E) Diagram connecting major screen hit pathways. Hits are colored by direction of phenotype; blue gene labels reduce translation disruption product accumulation, red gene labels increase translation disruption product accumulation.

Article Snippet: Genome-wide CRISPRi and CRISPRa sgRNA libraries (hCRISPRi_v2: Addgene #83969 and #83970; hCRISPRa_v2: #83978 and #83979) were amplified, packaged into lentiviral particles, and titers were determined as described in ref . For K562, CRISPRi parental cells (187 million) were infected at a multiplicity of infection (MOI) of 0.28 and selected using 2 μg/mL puromycin (Sigma) for 6 days starting 48 h post-transduction.

Techniques: Control, Disruption, Expressing, Genome Wide, Flow Cytometry, MANN-WHITNEY, Labeling

Journal: bioRxiv

Article Title: Ribosome-associated quality control of aberrant protein production during amino acid limitation

doi: 10.64898/2026.01.14.699605

Figure Lengend Snippet: (A) Schematic outlining how the screen with reporters and sorting scheme depicted in was performed. Cells were arginine limited for 3 days, sorted, recovered, and re-sorted into the same bin after a second period of arginine limitation. After recovery, guide RNAs were sequenced to calculate enrichment scores. (B,C) Western blot (B) and flow cytometry (C) validation of selected hits with negative or positive phenotype scores across various pathways in K562 cells. Cells expressing CRISPRi targeting hits (or non-targeting controls; NTC) and dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ) were arginine limited for 3 days to assess translation disruption product levels (B,C) and signaling responses through mTOR, GCN2 and ZAKα (B). In (B), “*” marks non-specific band from blot stripping and reprobing. (D) Western blot to assess translation disruption and GCN2 response in 293T cells overexpressing GADD34 or an NTC by CRISPRa (scFv-sfGFP-GCN4-VP64) and dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ), with or without limitation for arginine for 5 days and treatment with 40 nM ISRIB. (E) Western blot to assess translation disruption with or without limitation for leucine or arginine for 7 days and treatment with 250 nM Torin1 in MiaPaCa cells expressing the Flag-YFP CGG -2xAGA-DHFR CGG reporter. (F) Flow cytometry to assess translation disruption product accumulation upon limitation for arginine with or without GCN2 knockdown by CRISPRi and 250 nM Torin1 treatment in K562 cells expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ). (G-H) Western blots to assess phospho-p38 and -JNK response to arginine or leucine limitation and 0.1 μg/mL anisomycin treatment with or without treatment with 5 μM SB202190 (p38 inhibitor) in wild-type and GCN2 KO 293T cells. (I) Western blot to confirm ZAKα KO in wildtype and GCN2 KO 293T cells as indicated. (J-K) Western blots to assess phospho-p38 and -JNK response to 0.1 μg/mL anisomycin treatment or UV irradiation (J) or arginine or leucine limitation (K) in wild-type and GCN2 KO, ZAKα KO, and ZAKα+GCN2 double KO 293T cells as indicated. (L) Flow cytometry to assess reporter fluorescence upon limitation for arginine for 6 days with or without treatment with 5 μM SB202190 in wild-type and GCN2 KO 293T cells expressing the Flag-YFP CGG -4xAGA-DHFR CGG (“AGA”) or Flag-YFP CGG -4xCGG-DHFR CGG (“CGG”) reporter as indicated. (M) Flow cytometry to assess reporter fluorescence over time upon limitation for arginine with or without treatment with 5 μM SB202190 in K562 cells with (sgGCN2) or without (sgNTC) GCN2 CRISPRi knockdown expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ). (N) Change in translation disruption reporter (Flag-YFP CGG -4xAGA-DHFR CGG (“AGA”) or Flag-YFP CGG -4xAGA-DHFR CGG (“CGG”)) mRNA level upon arginine limitation for 3 days with or without treatment with 5 μM SB202190 in 293T cells. (O) Change in translation disruption reporter mRNA level (Flag-YFP CGG -4xAGA-DHFR CGG ) upon arginine limitation for 3 days with (sgLAMTOR2) or without (sgNTC) CRISPRi knockdown of LAMTOR2 in 293T cells expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ), relative to cells expressing control guide (sgNTC 1). (C,L,N,O) Error bars represent standard error of the mean of 3 replicates. (B,D,E, G-K) Full-length reporter product is indicated (α-GFP antibody used to detect YFP, α-vinc = α-vinculin).

Article Snippet: Genome-wide CRISPRi and CRISPRa sgRNA libraries (hCRISPRi_v2: Addgene #83969 and #83970; hCRISPRa_v2: #83978 and #83979) were amplified, packaged into lentiviral particles, and titers were determined as described in ref . For K562, CRISPRi parental cells (187 million) were infected at a multiplicity of infection (MOI) of 0.28 and selected using 2 μg/mL puromycin (Sigma) for 6 days starting 48 h post-transduction.

Techniques: Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Disruption, Stripping, Knockdown, Irradiation, Fluorescence, Control